人卵母细胞样细胞体内分化模型的建立

赋予抗TNF-α 单链抗体片段 (TNF-scFv) 对炎症组织的特异性,用一段来自人清蛋白 (HSA) 的柔性连接肽在基因水平上连接TNF-scFv和抗B型纤维连接蛋白 (B-FN) 的额外域B (ED-B) 的scFv L19,构建了抗TNF-α/抗ED-B单链双特异抗体BsDb,其中B-FN为炎症组织中特异表达的抗原。BsDb在毕赤酵母中获得了分泌表达,表达产物经鉴定和纯化制备后,进行了功能分析。结果表明,BsDb保留了其亲本抗体TNF-scFv和L19对抗原的免疫反应性,能够同时结合TNF-α和ED-B,并中和TNF-α的生理作用。而且,BsDb对抗原的亲和力及中和能力与大肠杆菌包涵体来源的亲本抗体相比显著增强。在小鼠佐剂型关节炎 (AIA) 模型中,BsDb能选择性地积累和保留于小鼠的炎症关节,并快速从血浆中清除。说明BsDb兼备炎症组织的特异性和正常组织的低毒性,在类风湿关节炎及其他慢性炎症性疾病的治疗上具有较大潜力。     

BMP9诱导人脐带间充质干细胞体内外成骨分化的作用研究

目的:探讨人骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)在体内外诱导人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)成骨分化的作用研究。方法:设立Ad-BMP9处理组和Ad-GFP对照组感染hUC-MSCs,两组细胞分别于3天、5天、7天进行ALP活性检测,14天后采用免疫组织化学染色检测骨钙素(Osteocalcin,OCN)、骨桥蛋白(Osteopotin,OPN)的表达情况,21天后茜素红染色检测矿化结节的形成;然后收集不同分组hUC-MSC用于裸鼠皮下注射成骨模型的建立,4周后取出离体骨进行Micro-CT扫描和分析,并进行H&E、Masson Trichrome、Alcain Blue染色。结果:BMP9处理组的ALP活性和矿化结节形成明显高于对照组,免疫组化染色结果显示BMP9诱导组的OCN、OPG的阳性表达明显高于对照组;裸鼠皮下注射成骨模型的观察结果显示,空白对照组没有形成肉眼可见的皮下包块,仅感染Ad-BMP9的hUC-MSCs能生成异位骨,且形成的异位骨骨量明显,骨密度平均值为396.05±0.60;H&E染色结果显示BMP9诱导生成的异位骨中形成部分成熟的骨基质和骨小梁,Masson Trichrome染色结果显示BMP9明显诱导hUC-MSCs的基质矿化作用,Alcain Blue染色结果显示BMP9明显诱导hUC-MSCs的软骨内成骨作用。结论:BMP9成功诱导人脐带间充质干细胞的体内外成骨作用,为临床骨组织工程的细胞疗法提供了明确的可行性。     

人胚胎干细胞高效分化为巨核细胞模型的建立

人胚胎干细胞具有无限增殖潜能,且能够分化为包括巨核细胞在内的大多数的成熟血细胞。因此,为解决临床治疗血小板来源不足而建立一种由人胚胎干细胞高效分化获得成熟巨核细胞的分化模型具有重要应用前景。该文利用人胚胎干细胞与m AGM-S3基质细胞系共培养进行早期造血前体细胞的定向分化,并在分化第14 d获得大量"卵石样"造血前体细胞(CD43+细胞、CD45+细胞)。将"卵石样"造血前体细胞分离后,用无血清培养基分阶段添加SCF、TPO、IL-6、IL-3、FLt-3、IL-11等细胞因子进行巨核细胞定向诱导分化。结果显示,在分化的第3 d,CD41a+细胞比例可高达48.5%,CD42b+细胞比例可高达30.0%。对分化第6 d的细胞进行Wright-Giemsa染色,可见形态典型的巨核细胞。综上所述,该研究初步建立了一种人胚胎干细胞高效分化为巨核细胞的模型,为体外获得大量巨核细胞奠定了基础。     

尿酸影响人骨髓间充质干细胞成骨分化过程中BMP-2 表达

目的:探讨在人骨髓间充质干细胞(h BMSCs)成骨分化过程中,不同浓度尿酸(UA)对骨形态形成蛋白-2(BMP-2)表达的影响。方法:以全骨髓贴壁培养法分离h BMSCs,将生长状态良好的第3代h BMSCs分为5组,分别为空白对照组(加入完全培养基)和成骨诱导组(加入成骨诱导液及含0 mmol/L、0.2 mmol/L、0.4 mmol/L、0.8 mmol/L尿酸的完全培养基)。连续干预诱导14d后,用倒置显微镜观察细胞形态的变化,通过观察茜素红染色情况及检测碱性磷酸酶(ALP)活性进行成骨情况的检测。RT-PCR技术检测各组细胞BMP-2 mR NA的表达情况。结果:第3代h BMSCs大多为形态单一的长梭形,呈旋涡状生长;干预诱导后的细胞逐渐变成不规则的立方形,局部形成团块状结节,以含尿酸浓度为0.8 mmol/L的成骨诱导培养基最为显著。连续干预14d后,空白对照组茜素红染色为阴性,而各成骨诱导组细胞茜素红染色结果为阳性,提示干预诱导后的细胞为成骨细胞。碱性磷酸酶活性随尿酸浓度的增加和干预时间的延长而增强(P<0.05)。RT-PCR检测结果显示,空白对照组无BMP-2 mR NA的表达。成骨诱导组随培养基中尿酸浓度的增加,BMP-2 mR NA表达逐渐增强,呈浓度依赖性(P<0.05)。结论:尿酸上调h BMSCs向成骨细胞分化过程中BMP-2 mR NA的表达。     

人髓样分化因子88的生物信息学分析

人髓样分化因子88(MyD88)属于Toll样受体和IL-1受体家族成员下游的炎症信号通路的中心因子。为了研究人MyD88的蛋白结构和功能,本文利用重要数据库进行了生物信息学分析。研究表明,人MyD88基因全长为5 670 bp,位于染色体3p22. 2,共编码296个氨基酸。人MyD88蛋白具有较高的保守性,其等电点为5. 64,不存在信号肽,并且含有2个苏木化位点、15个丝氨酸磷酸化位点、6个苏氨酸磷酸化位点和4个酪氨酸磷酸化位点。该蛋白主要定位在细胞核和细胞质中,而且在血液、脾脏和肺较多,其二级结构含有145个α-螺旋和30个β-折叠,拉曼图表明三级结构模型A是可信的。与人MyD88相互作用的蛋白主要是Toll样受体和白介素相关蛋白,并且参与炎症反应、细胞凋亡等重要的免疫过程。本研究为阐明人MyD88基因结构特点和生物学功能奠定了理论基础,也为其相关疾病诊断和治疗提供了新思路。     

人诱导多能干细胞来源功能性胰岛β细胞的体外定向分化方法的建立

1型糖尿病是由胰岛β细胞功能受损、胰岛素分泌不足所致,目前,主要通过外源性胰岛素补充来治疗,但外源性胰岛素无法精准调控血糖,严重低血糖可危及生命。胰岛移植是一种替代疗法,但面临器官供体不足和异种来源胰岛β细胞存在人畜共患病交叉感染风险的问题。因此,获得足量且安全的胰岛β细胞是1型糖尿病细胞治疗面临的难题。本研究旨在通过人诱导多能干细胞(human induced pluripotent stem cells, hiPSCs)在体外向胰岛β细胞分化,提供一种潜在的1型糖尿病治疗新策略。为实现这一目标,我们采用了结合2D和3D培养系统的分化策略,模拟胰岛β细胞的体内发育环境,并使用多种生长因子调节在胰腺发育和β细胞分化中发挥重要作用的关键信号包括Notch信号通路(Notch signaling pathway)、Wnt信号通路(Wnt signaling pathway)、TGF-β/Smad信号通路(TGF-β/Smad signaling pathway)等,在体外将hiPSC定向诱导分化至胰岛β细胞。结果显示,在2D、3D结合的培养条件下,分化过程中定型内胚层细胞,胰腺祖细胞,胰腺...     

枸杞多糖诱导人脐血间充质干细胞向神经元样细胞分化的实验研究

目的:探讨枸杞多糖诱导人脐血间充质干细胞(MSCs)向神经元样细胞分化的可行性及其机制。方法:无菌条件下收集正常足月儿的脐带血,经肝素抗凝,用相对密度1.077的淋巴细胞分离液分离脐血的单个核细胞,用低糖DMEM培养基进行培养和纯化扩增。选取第3代细胞进行诱导实验,当传代细胞长满瓶底的80%以上时,先用含15?S和10ng/ml bFGF的DMEM完全培养基预诱导24小时,然后用不含血清含1g/L枸杞多糖的DMEM培养基诱导,光镜下观察细胞形态,用免疫组化技术检测细胞Nestin和NF的表达。结果:预诱导后MSCs没有变化,而经枸杞多糖诱导4h后细胞即出现形态学上的改变,细胞变成不规则形,立体感增强,从胞体伸出突起。免疫组化检测显示,细胞Nestin、NF呈阳性。结论:人脐血间充质干细胞经枸杞多糖诱导可转化为神经元样细胞,其诱导机制可能与枸杞多糖的抗氧化作用有关。     

中药固真方对成骨样细胞UMR106增殖分化的影响

 中药固真方具有补肾益精、延缓衰老的作用 .为了进一步探讨其作用机理 ,研究观察了固真方对成骨样细胞 UMR1 0 6DNA合成、原癌基因 c- fos,c- ki- ras m RNA表达以及骨钙素 (osteocal-cin)基因 m RNA表达的影响 .结果表明 :固真方可明显促进 DNA合成 ,且在 1 0 -5稀释度时达高峰 ;增殖相关的原癌基因 (c- fos,c- ki- ras) m RNA表达增强 ;成熟成骨细胞特征性标志蛋白——骨钙素基因 m RNA表达增强 .结果提示 :中药固真方可能通过影响一些与增殖相关的原癌基因及骨钙素基因表达而促成骨样细胞 UMR1 0 6的增殖和分化成熟     

体外化学诱导人骨髓间充质干细胞分化为心肌样细胞

To investigate the potential of adult mesenchymal stem cells (hMSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure of 5-azacytidine in vitro. A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073 g/mL Percoll and cultured in the right cell culturing medium as previously described. The phonotypes of hMSCs were identified by flow cytometry. The stem cells were cultured in cell culture medium (as control) and medium mixed with 5-azacytidine (5-aza, 3, 5, 10 micromol/L) (n=5, respectively) for cellular differentiation. We examined respectively with immunohischemistry at 21 days of inducement on desmin, cardiac-specific cardiac troponin I (cTnI), GATA4 & connexin43. The ultrastructures of induced cells were examined by transmission electron microscope. The results indicated that the hMSCs showed a fibroblast-like morphology with vortex distribution in their peak propagation, and express high level of CD44 but negative for CD34 and CD45. 20%-30% cells grown after 5, 10 microl/L 5-aza treatment connected with adjoining cells and coalesced into myotube structures after 14 days. After 21 days of culturing, immunohistochemistry revealed expression of desmin, GATA4, cTnI and connexin43 in 5, 10 micromol/L showed positive, but no cardiac specific protein were found in neither 3 micromol/L nor in control group. The ratio of cTnI positive stained cells in 10 micromol/L group were higher than that in 5 micromol/L group (65.3+/-4.7% vs 48.2+/-5.4%, p<0.05). Electron microscopy revealed myofilaments were formed. The results indicated that purified hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration.     

海绵状的小肠粘膜下层促进成骨样细胞增殖分化

目的:观察人脂肪干细胞(hADSCs)诱导分化的成骨样细胞在海绵状的猪小肠粘膜下层(SIS)表面的生长情况,探讨三维立体海绵状的SIS能否促进成骨样细胞的增殖和分化.方法:采用物理和化学结合的方法将猪近段空肠制备成脱细胞的SIS,再将薄膜状的SIS经液氮低温研磨制成微粒,交联后采用冷冻干燥技术重塑形为海绵状的SIS;原代培养hADSCs,流式术检测表面抗原,诱导其成骨、成软骨、成脂分化并染色鉴定;将诱导的成骨样细胞与海绵状SIS复合培养,扫描电镜观察细胞形态;应用SIS材料浸提液培养成骨样细胞,MTT法检测细胞增殖情况,ALP活度检测成骨分化情况.结果:脱细胞的SIS未见有核物质,海绵状的SIS呈三维立体状,具有大量均匀一致的孔隙;原代培养的hADSCs表达干细胞相关抗原,并可分化为成骨样细胞,茜素红将钙结节染成紫红色.成骨样细胞与海绵状SIS复合培养后,细胞生长旺盛增殖能力强,ALP表达量明显增加.结论:海绵状的SIS具有均匀的三维孔隙,细胞相容性好,能明显促进hADSCs来源的成骨样细胞的增殖及成骨分化,可成为骨组织工程新型的三维立体天然生物衍生材料.     

Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cells derived from human follicular fluid to oocyte-like cell

Follicular fluid (FF) is essential for developing ovarian follicles. Besides the oocytes, FF has abundant undifferentiated somatic cells containing stem cell properties, which are discarded in daily medical procedures. Earlier studies have shown that FF cells could differentiate into primordial germ cells via forming embryoid bodies, which produced oocyte-like cells (OLC). This study aimed at isolating mesenchymal stem cells (MSC) from FF and evaluating the impacts of bone morphogenetic protein 15 (BMP15) on the differentiation of these cells into OLCs. Human FF-derived cells were collected from 78 women in the assisted fertilization program and cultured in human recombinant BMP15 medium for 21 days. Real-time polymerase chain reaction and immunocytochemistry staining characterized MSCs and OLCs. MSCs expressed germline stem cell (GSC) markers, such as OCT4 and Nanog. In the control group, after 15 days, OLCs were formed and expressed zona pellucida markers (ZP2 and ZP3), and reached 20–30 µm in diameter. Ten days after induction with BMP15, round cells developed, and the size of OLCs reached 115 µm. A decrease ranged from 0.04 to 4.5 in the expression of pluripotency and oocyte-specific markers observed in the cells cultured in a BMP15-supplemented medium. FF-derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in promoting the differentiation of these cells, which may give an in vitro model to examine germ cell development.     

In vitro differentiation of human amniotic fluid‐derived cells: Augmentation towards a neuronal dopaminergic phenotype

Amniotic fluid is known to yield a number of cell types which are multipotent, ethically derived, genetically stable, easily grown, expanded and possess favourable immunogenicity, which has resulted in an increasing interest for use in various neuronal disorders such as Parkinson's disease. The neuronal potential of cells derived from the adherent fraction of amniotic fluid, routinely taken by amniocentesis, are least explored. The aim of the present study was to investigate the capacity of these cells for neuronal and dopaminergic differentiation using in vitro differentiation protocols with canonical inductive factors not previously tested. To do this, samples derived from multiple donors were grown under four conditions: standard serum‐containing media, NB (neurobasal) media designed specifically for propagation and maintenance of neuronal cells, NB media with addition of retinoic acid and BDNF (brain‐derived neurotrophic factor) for NI (neuronal induction), and NB media with addition of FGF8 (fibroblast growth factor‐8) and Shh (sonic hedgehog) after NI. Our results showed the presence of multiple neuronal markers after growth in serum‐containing medium [TUJ1, MAP2, NF‐M, TH (tyrosine hydroxylase)], which was significantly up‐regulated after serum withdrawal in NB medium alone with induction of NeuN (neuronal nuclei) and NSE (neuron‐specific enolase). NI and DA.I (dopaminergic induction) was accompanied by further increases in expression and a distinct transition to a sustained neuronal morphology. Western blot analysis confirmed increasing TH expression and NURR1, expressed in base serum‐containing media, found to be down‐regulated after induction. In conclusion, these cells possess a highly favourable base neuronal and dopaminergic prepotential, which may easily be accentuated by standard induction protocols.     

Induced in vitro differentiation of neural-like cells from human amnion-derived fibroblast-like cells

There is growing evidence that the human amnion contains various types of stem cell. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In this study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into neural cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of neural cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that neuron specific enolase, neurofilament-medium, beta-tubulin isotype III, and glial fibrillary acidic protein (GFAP) showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that neuron specific enolase, GFAP and myelin basic protein (MBP) were present in HADFIL cells following the induction of differentiation, although one of the HADFIL cell populations showed a lower expression of GFAP and MBP. These results indicate that HADFIL cell populations have the potential to differentiate into neural cells. Although further studies are necessary to determine whether such in vitro-differentiated cells can function in vivo as neural cells, these amniotic cell populations might be of value in therapeutic applications that require human neural cells.     

Amniotic fluid stem cells to study mTOR signaling in differentiation

The protein kinase mTOR is the central player within a pathway, which is known to be involved in the regulation of e.g., cell size, cell cycle, apoptosis, autophagy, aging and differentiation. mTOR activity responds to many signals, including cellular stress, oxygen, nutrient availability, energy status and growth factors. Deregulation of this enzyme is causatively involved in the molecular development of monogenic human diseases, cancer, obesity, type 2 diabetes or neurodegeneration. Recently, mTOR has also been demonstrated to control stem cell homeostasis. A more detailed investigation of this new mTOR function will be of highest relevance to provide more explicit insights into stem cell regulation in the near future. Different cellular tools, including adult stem cells, embryonic stem cells or induced pluripotent stem cells could be used to investigate the role of mTOR in mammalian stem cell biology. Here we discuss the potential of amniotic fluid stem cells to become a promising cellular model to study the role of signaling cascades in stem cell homeostasis.     

Amniotic fluid stem cells to study mTOR signaling in differentiation

The protein kinase mTOR is the central player within a pathway, which is known to be involved in the regulation of e.g., cell size, cell cycle, apoptosis, autophagy, aging and differentiation. mTOR activity responds to many signals, including cellular stress, oxygen, nutrient availability, energy status and growth factors. Deregulation of this enzyme is causatively involved in the molecular development of monogenic human diseases, cancer, obesity, type 2 diabetes or neurodegeneration. Recently, mTOR has also been demonstrated to control stem cell homeostasis. A more detailed investigation of this new mTOR function will be of highest relevance to provide more explicit insights into stem cell regulation in the near future. Different cellular tools, including adult stem cells, embryonic stem cells or induced pluripotent stem cells could be used to investigate the role of mTOR in mammalian stem cell biology. Here we discuss the potential of amniotic fluid stem cells to become a promising cellular model to study the role of signaling cascades in stem cell homeostasis.     

调节表皮干细胞增殖和分化的信号通路

表皮干细胞能够维持正常表皮的新陈代谢、毛囊周期循环以及参与创伤情况下创面的修复,皮肤肿瘤的发生也与其密切相关。表皮干细胞的增殖和分化受到严格的调控,了解表皮干细胞增殖与分化的调控机制将有助于治疗脱发、创伤以及皮肤肿瘤等疾病。文章着重概述了Wnt和Bmp信号对于控制干细胞命运的重要作用。     

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:212–219, 2015     

体外诱导人羊膜上皮细胞分化为胰岛素分泌细胞的研究

目的:通过体外诱导分化实验,探讨人羊膜上皮细胞(hAECs)向胰岛素分泌细胞(ISCs)分化的能力。方法:采用胰蛋白酶消化法从人羊膜组织分离提取hAECs,用流式细胞仪和免疫细胞化学法进行鉴定。取第3代hAECs在含尼克酰胺和N2补充物的无血清培养基中诱导培养,分别于诱导不同时间采用免疫细胞化学法检测胰岛素和β2微球蛋白的表达,采用放射免疫法检测上清液中胰岛素含量,采用RT-PCR检测胰岛素mRNA和胰十二指肠同源异型盒因子-1(PDX-1)mRNA的表达。结果:①hAECs高表达CD29、CD73、CD166和CK19;②hAECs诱导组第7、142、1天胰岛素阳性细胞百分率分别为74.00%±1.73%、75.33%±1.15%和75.67%±0.58%,而对照组未见胰岛素阳性细胞;③hAECs诱导组第7、14、21天培养物上清液中胰岛素含量分别达(328.47±3.22)μIU/ml、(332.26±1.22)μIU/ml和(329.68±2.57)μIU/ml,均显著高于对照组(P均<0.01);④hAECs诱导前后均有PDX-1 mRNA和β2微球蛋白表达,胰岛素mRNA表达仅见于诱导组。结论:hAECs能分化为ISCs,在Ⅰ型糖尿病细胞移植治疗方面具有潜在应用前景。